Natural products continue to inspire novel chemistry and biology. The enediynes represent one of the most fascinating families of natural products for their unprecedented molecular architecture and extraordinary biological activities. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. In this application, w propose to mine the genomes of the Actinomycetale collection at The Scripps Research Institute for new enediynes. Our hypotheses are: (i) genome survey of our strain collection for the enediyne polyketide synthase (PKS) gene cassettes will allow us to identify potential enediyne producers, (ii) genome sequencing of the potential producers for enediyne biosynthetic gene clusters will allow us to predict the structural novelty of the new enediynes, and (iii) genetic manipulation and fermentation optimization of the most promising strains will allow us to produce and isolate the new enediynes. We have completed a genome survey of 3,500 strains from our Actinomycetale collection and identified 92 strains (hits) that can be phylogenetically grouped into 48 clades according to the enediyne PKS cassettes. Genome sequencing of representative hits from 28 clades confirmed that each clade encodes distinct enediyne biosynthetic gene clusters. The specific aims for this grant are: (i) genome sequencing of representative hits from all 48 clades to identify a total of 48 distinct enediyne gene clusters and prioritize the hits for enediyne discovery and (ii) genetic manipulation and fermentation optimization of the most promising hits to produce and isolate the new enediynes for structural characterization. Discovery of an enediyne is a significant event in natural products chemistry, biology, and drug discovery. We aim at discovering 6-12 new enediynes from the 92 hits, nearly doubling the inventory of enediyne natural products known to date. The outcomes of this application include (i) fundamental contributions to genome mining and metabolic pathway engineering for natural product discovery and production, (ii) discovery of new enediyne natural products to inspire chemistry, biology, and medicine, and (iii) new insights into enediyne chemistry and biosynthetic machineries. The long-term goal of our research is to understand at a molecular level how microorganisms synthesize complex natural products and to exploit this knowledge to discover novel natural products and engineer their analogues for drug discovery.